Proteomic interaction and genomic action determinations in the presence of associated redox state conditions

ABSTRACT

Genomic actions and/or proteomic interactions for pathophysiological processes and for physiological processes are determined at associated redox state conditions. Protein interactions are correlated with oxygen tensions. Identification of markers for disease including epitopes is effected in the presence of simulated redox state perturbations. Screening for previously unknown receptors and activating ligands is carried out in the presence of alteration of redox state.

TECHNICAL FIELD

This invention is directed to a method of establishing genomic actionmaps and/or proteomic interaction maps for comparison ofpathophysiological and physiological processes and/or to identifyproteins including epitopes and/or genes involved in or representingmarkers for a disease, drug reaction, neoplasm (tumor) or infectionand/or to correlate protein interactions and/or to identify previouslyunknown receptors and/or activating ligands therefor.

BACKGROUND OF THE INVENTION

Strategies for studying cellular function involved in disease statesgenerally rely on comparison of control and disease states. A number ofdifferent proteomic and genomic strategies, including differentialprofiling platforms and functional assays (e.g., interaction studies)are being routinely employed for this purpose. At the heart of theseassays is the assumption that they can sensitively and specificallydetect differences in expression or function in a full complement ofgenes or proteins and that they accurately simulate thepathophysiological processes under investigation. However, unrecognizedflaws in current screening methodology are that they are carried out inair and, furthermore, are not carried out in the presence ofperturbations that are characteristic of and specific to thepathophysiological processes or otherwise representative ofphysiological conditions, e.g., in the presence of nitric oxide (NO).Thus, current screening methodology lacks a level of validation andbiological significance. The actions and interactions determined are notcausally related to the pathophysiological processes.

SUMMARY OF THE INVENTION

This invention relies on the recognition by the inventor thatpathophysiological processes occur in the presence of redox state/NOperturbations that are characteristic of and specific to thepathophysiological processes and that physiological processes occur atassociated redox state conditions and that establishing a genomic actionmap and/or proteomic interaction map comparing genomic actions and/orproteomic interactions of pathophysiological processes and physiologicalprocesses at such redox state conditions would provide results that arecausally related to the pathophysiological processes and more accuratefor the physiological processes and responses different from where theaction and interaction maps are not established at such redox stateconditions.

Consider that different pathophysiological and physiological processesare associated with different enzymatic reactions which cause relatedredox states and that the related redox states cause responsiveexpressions of genes and expression and/or formation of relatedmolecules. We have shown that different redox state modifier moleculesmake a difference in function altering effects on proteins and geneexpression. For example, we have found that protein cysteine centers canprocess as many as six different redox state related modifications,namely —SH, SNO_(x), SSG, SOH, S(O)S and SO_(x), into distinctfunctional (e.g., transcriptional) responses.

The term “pathophysiological process” is used herein to mean disease,drug reaction, neoplasm or infection.

The term “the physiological process” is used herein to mean the absenceof disease, drug reaction, neoplasm and infection.

The term “redox state” is used herein to refer to the electronic balanceof a system including covalent modification by NO related species,oxygen related species, or metal ions or other modifications caused bychanges in O₂ concentration or concentration of NO related species. Theterm “NO related species” is used herein to mean NO_(x) where x is 1 or2, NO⁻ and NO⁺ and organic derivatives thereof including nitrites andnitrates. The term “oxygen related species” is used herein to mean O₂and reactive oxygen species, for example, superoxide, hydrogen peroxideor lipid peroxide.

The term “redox state perturbation” is used herein to mean redox statealteration from normal (i.e., from physiological state).

In an embodiment, denoted the first embodiment, the invention isdirected to a method of establishing a genomic action map and/orproteomic interaction map for comparison of pathophysiological andphysiological processes comprising: (a) determining genomic actionsand/or proteomic interactions for the pathophysiological process in thepresence of simulated redox state perturbation(s) that is characteristicof and specific to the pathophysiological process; (b) determininggenomic actions and/or proteomic interactions for the physiologicalprocess in the presence of redox state that is associated with thephysiological process; and (c) generating a genomic action map and/orproteomic interaction map from the determination of (a) that is moreclosely correlated with the pathophysiological process than if thedetermination of (a) were not carried out in the presence of simulatedredox state perturbation(s) that is characteristic of and specific tothe pathophysiological process, and from the determination of (b) thatis more closely correlated with the physiological process than if thedetermination of (b) were not carried out in the presence of redox statethat is associated with the physiological process. The genomic actionsand/or proteomic interactions determined in (a) and (b) are compared todetermine genomic actions and/or proteomic interactions or activity thatare causally related to the pathophysiological process.

The term “genomic action” is used herein to mean change in level ofexpression of genes or change in activity of gene products.

The term “genomic action map” is used herein to mean display of changesof genomic action.

The term “proteomic interaction” is used herein to mean change in levelof expression of proteins or change in the interaction between proteinsor change in the interaction between proteins and other molecules (e.g.,DNA, RNA, lipids) or change in the activity of proteins.

The term “proteomic interaction map” is used herein to mean display ofthe changes of proteomic interaction.

Redox state perturbations are caused, for example, by redox statemodifier molecules in concentration variation from physiological state,glucose concentration variation from physiological state and pHvariation from physiological state as determined in affected tissue orcell or in blood perfusing affected tissue, or the presence oftransition metal or other thiol chelating metal such as zinc or byalterations in any NADH ratio.

The term “simulated redox state perturbation(s) that is characteristicof and specific to the pathophysiological process” is used herein tomean carrying out the genomic action and proteomic interactiondeterminations for the pathophysiological process in the method of thefirst embodiment herein in the presence of redox state perturbationsthat would be present in vivo in the pathophysiological process.

The term “redox state modifier molecule” is used herein to mean agent ormolecule that affects redox state of cell, proteins, DNA or lipid,including covalent or coordinate modification of thiol or metal bydiatomic or triatomic ligand, e.g., NO or O₂ in concentration present inthe pathophysiological process.

The term “redox state that is associated with the physiological process”is used herein to mean carrying out the genomic action and proteomicinteraction determinations for the physiological process in the methodof the first embodiment herein in the presence of redox state affectingconditions that would be present in vivo in the physiological process.

The term “causally related” is used herein to mean involved causally inthe pathophysiological process or associated with the disease process inways that would affect the outcome of the pathophysiological process orenable monitoring or detection of the pathophysiological process.

A different embodiment of the invention herein, denoted the secondembodiment, is directed to a method of identifying target proteinsand/or genes related to a disease (i.e., the expression of which causesor results from the disease process) comprising challenging cellsinvolved in the disease with agent(s) to produce and identify redoxstate-related modifications of proteins and/or lipids that wouldsubsequently mediate protein modifications (e.g., by acting as signalingmolecules) or interact with proteins, that are characteristic of thedisease.

The term “target” is used herein to mean protein or gene involved in thedisease process.

The term “challenging cells involved in the disease” is used herein tomean exposing them to various redox perturbations that arecharacteristic of the disease which are different from the standardconditions used to evaluate interactions or changes.

In one case, the agent(s) constitute at least one redox-state modifiermolecule which is generated in vivo in the disease and affects the redoxstate of the cells involved in the disease, and the modifications areprotein-protein interactions obtained in cells involved in the diseasein the presence of the redox state modifier molecule agent(s).

Both the first and second embodiments are useful to identify targetsthat are components of signaling circuitry or which are perturbed indisease states.

Both the first and second embodiments are useful to identify biomarkersof disease states.

The determination of protein binding partners in one aspect of the firstembodiment or in one aspect of the second embodiment, is useful as astarting point for determining how to keep the partners apart as therapyfor a disease state or to inhibit their activity.

The use of simulated redox state perturbations for determinations ofgenomic actions and proteomic interactions for a pathophysiologicalprocess causes identification of different genes and proteins fromconventional determinations and induction of protein-proteininteractions not present in the determinations in the absence ofsimulated redox state perturbations or inhibition of protein-proteininteractions that are present in the absence of simulated redox stateperturbations and thereby provide results which are more accurate forpathophysiological process identification than are obtained inconventional determinations and permit new functional assignments andpredictions, and new opportunities for drug design. A reason for this isthat redox state perturbations regulate gene expression and proteininteraction. The use of redox state that is associated with aphysiological process for determinations of genomic actions andproteomic interactions for the physiological process can causeidentification of different genes and proteins from conventionaldeterminations which are carried out in room air, in complete absence ofnitric oxide (NO) and without regard for redox state and a basis forcomparison which can be different from that in a conventionaldetermination.

Another embodiment herein, denoted the third embodiment herein, isdirected at a method of correlating protein interaction(s) with oxygentension, comprising determining protein interaction(s) in the presenceof oxygen tension different from that in room air, that is at a Po₂ lessthan 150 mm Hg. When a set for protein is used for the determinationthat is associated with a physiological process, the method can be usedto identify normal protein functions. When a set of proteins is used forthe determination that is associated with a pathophysiological process,the method can be used to identify protein functions associated with thepathophysiological process.

The term “protein interaction” is used herein to mean the same as“proteomic interaction” which is defined above.

The term “oxygen tension” is used herein to mean concentration ofoxygen.

The term Po₂ is used herein to mean oxygen tension.

The term “set of proteins that is associated with a physiologicalprocess” is used herein to mean proteins expressed under suchconditions.

The term “set of proteins that are associated with a pathophysiologicalprocess” is used here to mean proteins expressed under suchpathophysiological condition or whose expression or activity is changed.

Another embodiment of the invention herein, denoted the fourthembodiment, is directed to a method of identifying a previously unknownreceptor or ligand, comprising measuring activation of receptor ororphan receptor in the presence of alteration of redox state.

Still another embodiment of the invention, denoted the fifth embodiment,is directed to a method of determining epitopes involved in and/orrepresenting markers of disease, comprising immunolabeling affectedtissue or cells in the presence of redox state perturbations that arecharacteristic of the disease.

DETAILED DESCRIPTION

We turn now to the first embodiment herein, that is the method ofestablishing a genomic action map and/or proteomic interaction map forcomparison of pathophysiological and physiological processes comprising:(a) determining genomic actions and/or proteomic interactions for thepathophysiological process in the presence of simulated redox stateperturbation(s) that is characteristic of and specific to thepathophysiological process; (b) determining genomic actions and/orproteomic interactions for the physiological process in the presence ofredox state that is associated with the physiological process; and (c)generating an interaction map from the determination of (a) that is moreclosely correlated with the pathophysiological process than if thedetermination of (a) were not carried out in the presence of simulatedredox state perturbation(s) that is characteristic of and specific tothe pathophysiological process, and from the determination of (b) thatis more closely correlated with the physiological process than if thedetermination of (b) were not carried out in the presence of redox statethat is associated with the physiological process. The genomic actionsand proteomic interactions determined in (a) and (b) are compared todetermine genomic actions and proteomic interactions that are causallyrelated to the pathophysiological process.

As indicated above, the method of the first embodiment herein differsfrom conventional methods in making determinations for genomic actionsand proteomic interactions for pathophysiological processes in thepresence of simulated redox state perturbation(s) that is characteristicof and specific to the pathophysiological process and in makingdeterminations for genomic actions and proteomic interactions forphysiological processes in the presence of redox state that isassociated with the physiological processes.

We turn now to a description of the method of the first embodimentexcept for the new features.

Protein-protein interactions are preferably determined using two-hybridsystems. In these systems, reconstitution into a hybrid caused byprotein-protein interaction of bait protein with prey protein ismonitored by activation of a reporter gene. Two-hybrid systems arediscussed, for example, in Nandabalan et al. U.S. Pat. No. 6,083,693;Klein et al. United States Statutory Invention Registration H1,892;LeGrain et al. U.S. Pat. No. 6,187,535; and Rain, J. -C., et al. Nature409, 211-215 (Jan. 11, 2001). The bait is a protein or proteins known tobe involved in the pathophysiological process for which thedetermination is being made. The prey can be constituted of all proteinsand genes expressed in cells of an affected tissue or body fluid or aselection therefrom. Other methods of determining protein-proteininteractions (e.g., as described in Zhu, H., et al., Science 293,2101-2105 (2001) and as described below) can also be used.

A two-hybrid system used in the Example I herein is a yeast two-hybridsystem. In the system used in the Example I herein, bait proteins arefused to one part of a yeast transcription factor and preys (in the caseof the Example I herein, a library made up of certain genes expressed inresponse to pathophysiological influence, which express encoded proteinsfor the determination) are fused to another part of a yeasttranscription factor. When the two parts come together because ofprotein-protein binding, there is a transcription reaction which isdetectable by color indication and/or growth.

Systems for determining protein-protein interactions which are usefulherein are also described in Fung, E. T., et al., Current Opinion inBiotechnology 12:65-69 (2001) and Delneri, D., et al., Current Opinionin Biotechnology 12:87-91 (2001).

Systems for determining protein interactions or activity include thosedescribed in Sakura, T., et al., Cell (1998), 573-585 and Hare, J., etal., Nature Medium, Vol. 5, 1241-1242 (1999). These involve a search foran orphan receptor or ligand where readout is measured by changes inintracellular second messenger such as calcium or G-protein activity.

Other systems for determining protein interactions or activity includethat described in Scherer, P., et al., Nature Biotechnology 16, 581-586(1998). This involves a search for new epitopes that would be availablethrough protein-protein interaction. While the new feature of simulatedredox state perturbation is discussed later, it is envisioned that redoxsignals are themselves endogenous ligands and/or create new proteincomplexes that are thereby activated. Such receptor complexes (proteinmodules) provide new epitopes that represent markers for a specificdisease process, e.g., following a redox challenge, a polyclonalantiserum is raised against surface protein followed by immunodepletionof antibodies from a non-exposed cell or tissue.

Systems for determining change in level of protein expression aredescribed in Fung, E. T., et al., Current Opinion in Biotechnology 12:65-69 (2001).

Systems for determining change in the interaction between proteins andother molecules (e.g., DNA, RNA, lipids) are described in Ren, B., etal., Science 290, 2306 (2000) and in Marshall H. and Stamler, J. S.,Biochemistry 40, 1688 (2001).

Methods for determining genomic actions include methods for assaying theexpression of genes in differential display, e.g., as described inZohlnhofer, D., et al., Circulation, 103, 1396-1402 (2001) and SAGEwhere levels of mRNA are quantified through hybridization or other meansof quantification, e.g., as described in Zhang, L., et al., Science Vol.276, 1268-1272 (1997).

We turn now to the new features of the first embodiment fordeterminations for pathophysiological processes, namely carrying out thedeterminations of the genomic actions and proteomic interactions in thepresence of simulated redox state perturbations (instead of in theabsence of simulated redox state perturbations, which is conventional).

We tun now to the simulated redox state perturbations. As indicatedabove, redox state perturbations can be caused, for example, by redoxstate modifier molecules in concentration variation from physiologicalstate, glucose concentration variation from physiological state, pHvariation from physiological state, and by presence of metal ions and byalterations in any NADH ratio. As indicated in the description of themethod of the first embodiment, the simulated redox state perturbationsused are those that are characteristic of and specific to thepathophysiological process for which a genomic action map and/orproteomic interaction map is being determined.

The redox state modifier molecules are those compounds produced in vivocharacteristic of the pathophysiological process, which because of theirpresence and/or concentration affect redox state. The redox statemodifier molecules are often produced in enzymatic reactions associatedwith the pathophysiological process. A wide variety of enzymes areassociated with pathophysiological processes. For example, certainoxidases are highly activated in inflammatory bowel disease, but not inatherosclerosis. A certain oxidase is responsible for bone resorptionand another for hypertension. A certain diaphorase controls the activityof p53-dependent apoptotic death cascades (implicated in cancers), butnot of p53-independent apoptotic mechanisms. Nitric oxide synthasecauses redox mediated damage in diabetes. Other enzymes involved indisease and other pathophysiological processes include oxygenases,peroxidases, reductases, transferases and dehydrogenases and the enzymesystems that control each of the kinds of enzymes named hereinbefore.Redox state modifier molecules generated by enzymes include, forexample, superoxide, peroxides (e.g., hydrogen peroxide), alkoxides,sulfoxides, brominating species, chlorinating species, nitrosatingmolecules (e.g., NO and RSNO where R is, for example, amino acid,peptide or protein), and nitrating molecules (e.g., peroxynitrite) andNO generating molecules (e.g., Angeli's salt). These are generatedrelatively specifically in different diseases to different extentsand/or in different subcellular compartments and the means exist tomeasure these (with standard spectroscopic, immunological,electrochemical, chemical and photolytic approaches). Important redoxstate modifier molecules are reactive oxygen species including hydrogenperoxide and reactive nitrogen species including nitric oxide. Othersinclude enzymes that regulate glutathione, NADH and flavin levels andwhose activities can be pharmacologically or genetically altered.Another important redox state modifier molecule is O₂ in concentrationin affected body tissue. Body tissue oxygen concentrations are muchlower than the concentration of oxygen in air, room air having a Po₂ of150 mm Hg. For example, tumors can have a Po₂ in the range of 10 mm Hgand a Po₂ in the case of oxygen induced reperfusion can be 80 mm Hg.

In many cases, redox state modifier molecules and concentrations thereofassociated with a particular pathophysiologic process are already known.For example, superoxide is associated with the classical model of1-metal-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced Parkinson'sdisease and nitration is implicated in depletion of dopamine.Hypotensive shock is produced by NO produced by upregulation of nitricoxide synthase. In the cases where these need to be determined, this canbe accomplished as follows: In one method, a model of thepathophysiological process (e.g., a model of injury) is established,then potential redox state modifier molecules are identified, e.g.,after identifying redox state related enzymes (i.e., enzymescharacteristic of the pathophysiological process that may produce andparticipate in production of redox state modifier molecules), and thenthe redox state modifier molecules mediating the pathological processare determined (e.g., by establishing that inhibition of production ofthe redox state modifier molecules protects cells from injury). Theultimate object, of course, is to establish the consequence of the redoxstate modifier molecules on genomic and/or proteomic interactions.

We turn now to an example of redox state modifier molecule that producesredox state perturbation. Hyperglycemia (30 millimolar D-glucose)produces selective oxidative stress within the mitochondria. The sourceof this redox state perturbation is nitric oxide synthase and themolecule it produces in this case is superoxide.

For the simulated redox state perturbations, the enzymes responsible forgenerating the redox state modifier molecules can be used in place ofthe redox state modifier molecules.

The redox state modifier molecules and appropriate concentrationsthereof are used in the methods herein by addition of the molecules tothe experiment or by controlling the environment of the determination.

We turn now to the case where glucose concentration variation fromphysiological state causes redox state perturbation characteristic ofand specific to a pathophysiological process and the genomic actionand/or proteomic interaction determination for the pathophysiologicalprocess is carried out in the presence of such glucose concentrationvariation. Pathophysiological processes known to involve glucoseconcentration variation from non-pathophysiological state includediabetes and ischemia reperfusion injury. Where a pathophysiologicalprocess is not known to involve glucose concentration variation fromnon-pathophysiological state, this can be screened for by determinationof intracellular glucose and/or glycogen and/or by assessing whetherrespiration is aerobic or anaerobic (more glucose consumption).

Appropriate glucose concentration where this causes redox stateperturbation characteristic of and specific to a pathophysiologicalprocess is imparted in the methods herein, for example, by addingglucose to cells, e.g., to provide a concentration of 30 millimolarD-glucose or restricting glucose or regulating oxygen concentration orregulating aspects of tissue metabolism.

We turn now to the case where the redox state perturbations involve pHvariation from non-pathophysiological state. In some cases,pathophysiological process are known to involve pH variation. Forexample, pH variation is associated with mitochondria during apoptosis,with ischemic areas and with abscess or infected area. Where apathophysiological process is not known to involve pH variation fromnon-pathophysiological state, this can be screened for by dyes orelectrodes. Appropriate pH where this causes redox state perturbationcharacteristic of and specific to a pathophysiological process isimparted in the methods herein, for example, by changing the pH of themedium used for the determination or by uncoupling mitochondrialrespiration.

We turn now to the case where the redox state perturbation involvesaddition of metal ions. Disorders are known which are associated withalteration of metal presence from normal. For example, acrodermatitisenteropathatica results from malabsorption of zinc, and Wilson's diseaseinvolves copper toxicosis. There is a secondary effect of redox stateperturbation where the alteration of metal amount affects redox state.For example, copper ions are well known to participate in redoxreactions and zinc and cadmium influence the redox state of cells, e.g.,by chelating thiol. Appropriate metal ion concentration can be effectedby addition of appropriate metal ion to determination medium.

We turn now to the case where redox state perturbation involvesalteration of an NADH ratio. NADH concentration is altered, e.g., in thecase of sleep disorders related to circadian rhythms. Alteration of NADHlevel can be produced, for example, by knockout of lactate dehydrogenase(LDH), e.g., in yeast cells in a yeast two-hybrid determination.

Following is an application of the first embodiment to determiningprotein-protein interactions for a pathophysiological process. Thepathophysiological process chosen for illustration is interstitialpulmonary fibrosis. To identify pertinent redox state modifiermolecules, redox-related enzymes responsible for generation of reactiveoxygen and/or reactive nitrogen species in pulmonary fibroblast cells,e.g., in response to platelet derived growth factor causing fibrosis,are identified. Based on the enzymes, the putative redox state modifiermolecules are identified. From these the redox-state modifier moleculesthat cause injury and/or the type of protein modification that is mostprevalent, are determined. Any protein known to be involved infibroproliferative disorders in the lung is used as bait. All proteinsor genes expressed in pulmonary fibroblast cells (cDNA library) are usedas prey. As a further measure of specificity, only those genes orproteins that are highly expressed in fibroproliferative disorders, asidentified by differential profiling, are utilized. A yeast two-hybridsystem determination is carried out at body oxygen concentration asdetermined in pulmonary fibrosis tissue with exposing of the system tothe redox state modifier molecules identified above as causing injuryand/or the type of protein modification that is most prevalent, toidentify new binding partners, and/or inhibition of other binding, andthus new drug targets. Alternatively, antibodies are generated to cellepitopes before and after production of reactive oxygen species and newepitopes are thereby discovered.

Following is another example of application of the first embodiment todetermining protein-protein interactions for a pathophysiologicalprocess. Having determined that mitochondrial nitric oxide synthasemediated superoxide production is the source of redox state perturbationin hyperglycemia, endothelial cell mitochondrial protein-proteininteractions are determined in the presence of superoxide generatingsystems, e.g., by adding paraquat or by transfecting yeast with nitricoxide synthase and then adding paraquat.

We turn now to the new features of the first embodiment fordeterminations for physiological processes, namely carrying out thedeterminations of genomic actions and proteomic interactions in thepresence of redox state that is associated with the physiologicalprocess.

The proteins for which the interactions are determined are any that areexpressed in the physiological process and can be, for example,expressed in the kind of tissue that is affected by thepathophysiological process compared to, and/or can be the same proteins(e.g., the same baits and preys) as used in the determination for thepathophysiological process compared to. As indicated, the determinationsof genomic actions and proteomic interactions for the physiologicalprocesses are carried out in the presence of redox state condition thatis associated with the physiological process; such redox state conditionincludes physiological Po₂, physiological concentrations of NO,physiological levels of nitrosothiols, very low levels of reactiveoxygen species, and reducing conditions. We turn now to physiologicaloxygen concentrations. The oxygen levels utilized are much lower thanthe current level which is conventionally used, namely the concentrationof oxygen in air, room air having a Po₂ of 150 mm Hg. The oxygenconcentrations utilized are preferably those in the tissue or organ orblood perfusing therethrough for the physiological process. This varieswidely. For example, alveolar Po₂ is 100 mm Hg, skeletal muscle Po₂ranges from 10 to 30 mm Hg and exercising muscle is still lower and thePo₂ in the villus (a loop in the small intestine) is close to zero.Moreover, while physiological Po₂ is considered to be 30 mm Hg, the Po₂on running is 5 mm Hg and rises above 30 mm Hg on stopping of running,and the Po₂ in the brain associated with thinking is 10-20 mm Hg. Thus arange of oxygen concentrations are considered suitable for thephysiological determination but the oxygen concentration used should belower than that of room air and preferably is below 100 mm Hg. Thephysiological concentration of NO utilized is nanomolar to submicromolarconcentration. The physiological levels of nitrosothiols utilized range,for example, from 1 nM to 10 μM. The physiological levels of reactiveoxygen species utilized range, for example, from 10⁻¹⁰M to 10⁻⁶M. Weturn now to the reducing conditions. Reducing conditions are not presentin conventional determinations which are carried out in room air.Reducing conditions can be provided, for example, by adding thiols orNAD(P)H, lowering O₂ concentration, adding chelating metals, addingphysiological levels of ascorbate, e.g., to provide a concentration of100 μM, or adding vitamin E. The appropriate redox state conditions canbe effected by adding molecules to the experiment or by controlling theenvironment of the determination.

We turn now to the second embodiment herein, i.e., the method ofidentifying target proteins and/or genes in a disease specific mannercomprising challenging cells involved in a disease with agent(s) toproduce redox state-related modification of proteins and/or lipids thatwould subsequently mediate protein modification or provide interactionswith proteins, that are characteristic of the disease.

The method of the second embodiment can be carried out, for example,using high throughput screens for proteins, e.g., as described in Fung,E. T., et al., Current Opinion in Biotechnology 12, 65-69 (2001) andcomputer based bioinformatic approaches as described in Fung, E. T., etal., Current Opinion in Biotechnology 12, 65-69 (2001) in the presenceof the agent(s) to produce redox state-related modifications of proteinsand/or lipids that are characteristic of the disease, to identifyspecific redox state-related modifier molecules and specific protein andlipid related changes and thereby create redox maps of disease. Suchmaps can be used to create redox chips that are specific for diseasessuch as atherosclerosis or Alzheimer's disease where the samples used inconjunction with the chips can be DNA or RNA or protein material. Theagent(s) to produce redox state related modifications are, for example,the redox state modifier molecules described above. Preferably, thesecond embodiment is carried out in the presence of oxygen concentrationas determined in the tissue or organs affected by the disease or inblood perfusing said organs.

We turn now to the method of the third embodiment herein, that is themethod of correlating protein interaction(s) with oxygen tension,comprising determining protein interaction(s) in the presence of oxygentension different from that in room air, i.e., in the presence of oxygentension less than 150 mm Hg.

The method of the third embodiment can be carried out using theconventional methods for determining protein-protein interactions, forexample, two-hybrid systems, including yeast two-hybrid systemsdescribed above, except that the determinations are not carried out inroom air but in the presence of oxygen tension less than that in roomair, i.e., at Po₂ less than 150 mm Hg.

The set of proteins utilized for the third embodiment is preferably aset of proteins associated with a physiological process or apathophysiological process. The term “set of proteins associated with aphysiological process” is used herein to mean proteins expressed orknown to be involved in the physiological process. Examples of sets ofproteins associated with a physiological process are ryanodine receptorin the case of force producing in the heart or NMDA receptor in the caseof normal cognition. The term “set of proteins associated with apathophysiological process” is used herein to mean proteins whoseactivity or expression changes in such a process. Examples of sets ofproteins associated with a pathophysiological process are NMDA receptorin stroke or caspase 3 in apoptosis.

The oxygen tensions used in the third embodiment preferably range from0.1 mm Hg to 145 mm Hg, e.g., from 5 mm Hg to 100 mm Hg.

Preferably, a plurality of determinations are carried out for each setof proteins, with different oxygen tensions being employed in eachdetermination, e.g., using 5 or 10 different oxygen tensions where theoxygen tensions employed are in increments of 5 or 10 mm Hg.

Where the set of proteins used is one associated with a physiologicalprocess, the method of the third embodiment herein is useful, forexample, to identify normal protein functions.

Where the set of proteins used is one associated with apathophysiological process, the method of the third embodiment herein isuseful, for example to identify protein fictions associated with thepathophysiological process.

We turn now to the fourth embodiment herein, which is a method ofidentifying previously unknown receptor or orphan receptor or activatingligand therefor comprising measuring activation of receptor or orphanreceptor in the presence of alteration of redox state of ligand. Aspreviously indicated, general methods for identifying receptor or orphanreceptor and activating ligand are described in Sakura, T., et al.,Cell, 573-585 (1988) and Hare, J., et al., Nature Medium, 5, 1241-1242(1999). This class of method is modified in the invention herein byscreening for receptor or orphan receptor or activating ligand bycarrying out the identifying methods in a series of runs in the presenceof a series of redox state perturbations, whereby the receptor andactivating ligand are associated with particular redox stateperturbations.

We turn now to the fifth embodiment herein, which directed to method ofdetermining epitopes involved in and/or representing markers of disease,comprising immunolabeling affected tissue or cells in the presence ofredox state perturbations that are characteristic of the disease.General methodology useful in this method is described in Scherer, P.,et al., Nature Biology 16, 581-586 (1998). The method of Scherer et al.is modified in the invention herein, in carrying out the determinationin the presence of redox state perturbations that are characteristic ofthe disease.

The invention is illustrated in the following working example.

EXAMPLE I

Macrophages exposed to cytokines (tumor necrosis factor andinterleukin-1), a well established model injury that is apoptotic innature, were used.

Potential redox state-related modifier molecules were determined asbeing nitric oxide, superoxide and hydrogen peroxide, on the basis thatthese were detected as being produced in high levels in macrophagesexposed to cytokines.

It was then established that nitric oxide but not superoxide or hydrogenperoxide is causal to cell injury by demonstration that inhibitors ofnitric oxide protected the cells but inhibitors of superoxide orhydrogen peroxide did not.

We then determined that nitric oxide, in addition to causing cellinjury, also inhibited proteins involved in protection againstcytokines. This indicates that non-specific binding of nitric oxide islikely to have some deleterious consequences. We further measuredmany-fold increased levels of S-nitrosothiol proteins. This indicatesthat what is needed is a way to identify the S-nitrosylated proteinsthat are the targets of nitric oxide and/or the functional consequencesof these modifications, so these modification can be manipulated withoutinhibiting proteins that protect against cytokines. Thus, the goal is toidentify the S-nitrosylated proteins that are the targets of nitricoxide and/or the functional consequences of modifications mediatedthereby.

For this purpose, protein-protein interactions were determined in ayeast two-hybrid system by the method described in Uetz, P., et al.,Nature 403, 623-627 (2000) and Ito, T., et al., DNAS, 97, 1143-1147(2000). The baits were all known proteins involved in apoptoticcascades. The preys were a library of macrophage genes (mRNA) expressedin response to cytokines that induce apoptosis. Mating pairs ortransfectants were exposed to continuous nitric oxide presence at levelsand flux rates mimicking those which are generated in macrophages, over18 hours (the time course, over which apoptosis occurs in these cells).Room air was used in the determinations to mimic the cell condition atfirst and subsequently low Po₂ (about 5 mm Hg) was used to improvespecificity and increase yield. New partners and/or inhibition ofpartners that were otherwise present, were sought. When caspase was usedas a bait, it was found that nitric oxide induced a novelapoptosis-inducing interaction (based on sequence of the interactingprotein) thus revealing a new and specific approach to inhibitingapoptosis (i.e., drugs that would inhibit interaction between caspaseand the new protein). When a second protein was used, nitric oxideinduced an interaction with a different protein. Use of other baitsresulted in determination of further interactions.

Comparison is to determination for a physiological process using thesame baits and preys in a yeast two-hybrid system using the redox stateconditions, room air, nanomolar to submicromolar concentration of nitricoxide, nanomolar to micromolar level of nitrosothiols, no reactiveoxygen species and reducing conditions provided by glutathione and NADH.

In all, 18 new targets were identified in response to nitric oxide, andone was identified by lowering the Po₂.

The presence of hydrogen peroxide in the determinations did not produceor modify the majority of determined interactions indicating use of bodyoxygen concentration instead of room air would not have made asignificant difference in those cases and shows specificity fordifferent redox modifiers.

EXAMPLE II

Bovine endothelial cells are exposed to LDL cholesterol to induceatherosclerotic changes including production of reactive oxygen species.The predominant reactive oxygen species are identified as beingsuperoxide and hydrogen peroxide, and used in a yeast-2 hybriddetermination at Po₂ of about 70 to identify novel interacting partners,of which one is shown to cause cell death. Inhibiting that new proteinis shown to be anti-atherogenic.

EXAMPLE III

Human umbilical vein endothelial cells are exposed to 30 millimolarD-glucose which impairs endothelial cell function as measured by loss ofnitric oxide bioactivity. Increased oxidative stress is localized to themitochondria using a fluorescent dye and superoxide is shown to be theoxidant (causing the oxidative stress). A yeast two-hybrid determinationusing multiple mitochondrial proteins identifies novel interactions inthe presence of superoxide generated by depleting cells of arginineand/or adding paraquat as compared to determination without superoxidebeing present. In addition, new surface epitopes are found to be presentin cells as identified by antibodies and as described in methodsdescribed above.

EXAMPLE IV

Macrophages exposed to TNF and interferon gamma were kept at Po₂ of 5 mmHg. Protein expression was compared in 2D-gels (by differentialprofiling) to Po₂ of 100 mm Hg. A yeast 2 hybrid was then establishedfor the proteins only expressed at low Po₂, showing previouslyunappreciated interactions.

Variations

Many variations will be obvious to those skilled in the art. Therefore,the invention is defined by the claims.

1. A method of correlating protein-protein interaction(s) involved inone or more pathophysiological processes or one or more physiologicalprocesses with oxygen tension comprising (a) screening for aprotein-protein interaction between at least one protein and a pluralityof proteins, where the screening is performed in room air, and where theplurality of proteins are screened concurrently; (b) screening for aprotein-protein interaction between the at least one protein and aplurality of proteins, where the screening is performed in the presenceof decreased oxygen tension from that in room air, where the oxygentensions employed in step (b) range from 0.1 mm Hg to 145 mm Hg, andwhere the plurality of proteins are screened concurrently; and (c)correlating the protein-protein interaction(s) with oxygen tension byidentifying at least one different protein-protein interaction between(a) and (b), wherein the at least one different protein-proteininteraction between (a) and (b) is involved in one or morepathophysiological processes or one or more physiological processes. 2.The method of claim 1 where a plurality of determinations are made instep (b) with different oxygen tensions being employed in eachdetermination.
 3. The method of claim 1 where the different interactionsin step (c) are used to identify protein functions associated with apathophysiological process.
 4. A method of correlating protein-proteininteraction(s) with oxygen tension comprising: (a) screening for aprotein-protein interaction using a yeast two hybrid system between atleast one protein and a plurality of proteins, where the screening isperformed in room air, and where the plurality of proteins are screenedconcurrently; (b) screening for a protein-protein interaction using ayeast two hybrid system between the at least one protein and a pluralityof proteins, where the screening is performed in the presence ofdecreased oxygen tension from that in room air, where the oxygentensions employed in step (b) range from 0.1 mm Hg to 145 mm Hg, andwhere the plurality of proteins are screened concurrently; and (c)correlating the protein-protein interaction(s) with oxygen tension byidentifying at least one different protein-protein interaction between(a) and (b).
 5. The method of claim 4 where the at least one protein isassociated with a physiological process or a pathophysiological process.6. The method of claim 4 where a plurality of determinations are made instep (b) with different oxygen tensions being employed in eachdetermination.
 7. A method of correlating protein-protein interaction(s)with oxygen tension comprising (a) screening for a protein-proteininteraction between at least one protein and a plurality of proteins,where the screening is performed in room air, and where the plurality ofproteins are screened concurrently; (b) screening for a protein-proteininteraction between the at least one protein and a plurality ofproteins, where the screening is performed in the presence of decreasedoxygen tension from that in room air, and where the plurality ofproteins are screened concurrently; (c) correlating the protein-proteininteraction(s) with oxygen tension by identifying at least one differentprotein-protein interaction between (a) and (b) and wherein a pluralityof determinations are made in step (b) with different oxygen tensionsbeing employed in each determination.
 8. The method of claim 7 where aplurality of determinations are made in step (b) with different oxygentensions being employed in each determination.
 9. The method of claim 7where the oxygen tensions employed in step (b) range from 0.1 mm Hg to145 mm Hg.